Ergosterol peroxide, with the full name of 5α,8α-Epidioxyergosta-6,22-dien-3β-ol, is a known compound, belonging to the sterols, widely existing in edible-medicinal fungus or other fungus. There are many reports about its activities; for example, Chinese patent application No. 201410311586.3 discloses that it has synergy with taxol in killing or damaging Hela cancer cells, reducing the side effects of chemotherapy and its cost by reducing the necessary dosage of taxol. Chinese application No. 200610126091.9 discloses that ergosterol peroxide has significant anti-tumor (human breast cancer and liver cancer) pharmacological activity, and has no harmful effects on immune organs and weight. Chinese application No. 201210233192.1 reported that it has significant inhibitory effect on malignant breast cancer cells (MT-1), lymphoma cancer cells (Jurkat) and brain tumor cells (U87), and kills drug-resistant brain tumor stem cells. Chinese application No. 200810119948.3 reported that ergosterol peroxide has remarkable antifungal bioactivity and can be used in the field of natural antibiotics. Chinese application No. 01817014.5 disclosed that ergosterol peroxide has strong inhibitory effects on the formation of melanin and can be used in the field of whitening cosmetics. Chinese application No. 01817014.5 disclosed that ergosterol peroxide has poisoning effects on agricultural pests, such as pieris rapae, prodenia litura, aphid and so on, and is therefore applicable to the field of pesticides. In addition, ergosterol peroxide is reported in other literature to have extensive pharmacological actions, such as antioxidation, antituberculosis, anti-inflammation, anti-atherosclerosis, inhibiting the proliferation of T-cells, and so on.
There are fewer reports on the methods of extraction, separation and purification of ergosterol peroxide, which was identified from isolated unknown monomers in most reports, and in a fraction of these reports some authors still misjudge it altogether. Chinese application No. 201210233192.1 broke the sporoderm of Ganoderma lucidum spore powder by the enzyme method and obtained the extract by the supercritical carbon dioxide extraction method. Tracing activity by the tumor inhibitory rate, a single monomer with highest activity, which was identified as ergosterol peroxide, was obtained by three times positive silica gel column chromatography with chloroform, ethyl acetate, petroleum ether-ethyl acetate as eluent in sequence, and followed by preparative chromatography. Using Naematoloma fasciculare fruit body and mycelium as materials, Chinese application No. 201210233192.1 disclosed obtaining two monomers by extracting with methyl alcohol and petroleum ether in turn, positive silica gel column chromatography with cyclohexane-ethyl acetate as eluent, and recrystallizing several times; one of the monomers was identified as ergosterol peroxide. In Chinese application No. 200810119948.3, a petroleum ether fraction from the fermented liquid of fungus Ppf4 and another petroleum ether fraction from the acetone extract of hypha were merged; three monomers were obtained by positive silica gel column chromatography with cyclohexane-acetone as eluent, followed by gel column and reversed silica gel column chromatography; one of the monomers was identified as ergosterol peroxide. Using the fruit body of Ganoderma lucidum as material, Cheng Chunru (“Chemical constituents from the fruiting body of Ganoderma lucidum with cytotoxicity investigations”, Journal of Shenyang Pharmaceutical University, 2014, 31(2): 102-106) obtained a fraction by extraction with 95% ethanol, extraction with petroleum ether, and positive silica gel column chromatography with petroleum ether-ethyl acetate as eluent; removing a monomer with recrystallization, another monomer was obtained finally by semi-preparative isolation from the mother liquid and identified as ergosterol peroxide. Using Shiraia bambusicola as material, Liu Yafeng, et al. (“Separation of peroxy-ergosterol in fungus” Zhuhuang, Journal of Tianjin University of Traditional Chinese Medicine, 2004, 23(1): 15-16) obtained a monomer, which was identified as ergosterol peroxide, by extracting several times with ethyl acetate and positive silica gel column chromatography. Zhang Nengsheng et al. (“Isolation and Purification of Peroxy-ergosterol from Paecilomyces fumosoroseus by High-Speed-Counter-Current Chromatography and identification by ESI-MS”, Food and Fermentation Industries, 2009, 35(6): 14-16) studied the isolation and purification of peroxy-ergosterol from paecilomyces fumosoroseus by advanced high-speed-counter-current chromatography, but the obtained target product is actually not peroxy-ergosterol, because the obtained target has no UV absorption at 240-400 nm, while 280 nm was used as UV absorption wavelength to detect the process of isolation and to test the purity in the report, and the chromatogram showed a larger absorbency. The author may have made a mistake.
The above-mentioned techniques of separation and purification of ergosterol peroxide have something in common: extraction with neutral or strong polarity organic solvent at first, then removing impurities by extraction for several times with weak polarity organic solvent, followed by several rounds of positive silica gel column chromatography repeatedly with weak polarity organic solvent having a certain ratio as eluent, collecting the target fractions, and then obtaining the monomer of ergosterol peroxide by the steps of gel chromatography, preparative chromatography, recrystallization and so on; the yields of ergosterol peroxide are mostly in milligrams, and the processes started without a known destination, that is, the monomer was obtained at first, and then was identified as ergosterol peroxide later. Preparation was meant to discover the chemical composition of natural plants and fungi without an intended definite end result; accordingly, the preparation process proved complex and varied with little consideration for cost and yield. Furthermore, the preparation process involved toxic reagents without regard for environmental safety. In summary, the above mentioned techniques are not suitable for direct application in industrial production.
The simulated moving bed chromatography separation system is the organic combination of simulated moving-bed technology and chromatographic separation technology. The efficiency of a moving bed is simulated by adopting chromatographic columns in serial connection while an electromagnetic valve permits continuous switching between inlet and outlet, allowing the stationary phase to circulate with a continuous countercurrent. The combination of simulated moving-bed and chromatography changed chromatographic separation from a discontinuous into a continuous process, allowing large output, high yield, and high purity, and has been applied in the field of petroleum and chemicals, food, fermentation, medicine and so on, mainly in the purification of petrochemicals, saccharide, fermented product, active components in traditional Chinese medicine, and chiral compounds. But there are yet no reports about purification of ergosterol peroxide by using simulated moving-bed technology.